The present invention relates generally to biocompatible materials. In particular, the present invention relates to cytomimetic systems having stabilized, phosphatidylcholine-containing polymeric surfaces. The biomaterials produced in accordance with the invention comprise various modular surface designs and have various applications such as in medical devices, vascular grafts, surgical equipment, drug delivery systems, etc.
The ability to repair, reconstruct and replace components of the human cardiovascular system is dependent upon the availability of blood compatible biomaterials. Biocompatibility refers to the interactions of living body tissues, compounds and fluids, including blood, etc., with any implanted or contacting polymeric material (biomaterial). Biocompatible biomaterials are of great importance in any biomedical application including, for example, in the implantation of vascular grafts and medical devices such as artificial organs, artificial heart valves, artificial joints, synthetic and intraocular lenses, electrodes, catheters and various other prosthetic devices into or on the body. Such applications, however, have been hampered by the lack of suitable synthetic materials that are stable when contacted with physiological fluids, particularly blood.
Exposure of synthetic biomaterials to body fluids such as blood, for example, can result in adverse reactions such as the formation of thrombi due to deposition of blood proteins (e.g., albumin, immunoglobulins, etc.) and/or adsorption of cell adhesive proteins (e.g., fibrinogen, fibronectin, vitronectin, etc.) causing platelet adhesion, activation and aggregation, as well as activation of the clotting cascade. Additionally, immune complexes can develop and stimulate undesirable immune responses such as proteolysis, cell lysis, opsonization, anaphylaxis, chemotaxis, etc.
Several approaches have been proposed for improving the biocompatibility of biomaterials useful in medical applications. For example, modifying the biomaterial surface to provide either low polarity or ionic charge or coating the surface with biological substances such as cells, proteins, enzymes, etc. has been used to prevent undesirable protein adhesion. Another approach involves the incorporation of an anticoagulant into the biomaterial, rendering the biomaterial antithrombogenic. A further approach involves the incorporation of various phospholipids into the biomaterial. An additional approach involves the binding of a heparin-quaternary amine complex, or other antithrombotic agent, to the biomaterial surface. However, many of these methods have the disadvantage of being nonpermanent systems in that the surface coating is eventually stripped off or leached away. For example, heparin, which is complexed to the biomaterial surface, is not only gradually lost from the polymer surface into the surrounding medium but also has limited bioactivity due to catabolism and its inherent instability under physiological conditions.
Thus, a need still exists for a biocompatible material for use in various medical applications possessing desired physical and surface characteristics and also exhibiting antithrombogenic properties.
One of the most intriguing developments in the past decade has been the recognition that membrane-mimetic systems having a phosphorylcholine component limit the induction of surface-associated blood clot formation. This biological property has been attributed to the large amount of surface bound water due to the zwitterion structure of the phosphorylcholine head group. It has also been suggested that specific plasma proteins which inhibit the blood clotting process are selectively adsorbed to this head group (Chapman [1993] Langmuir 9:39).
Natural membranes are utilized as models for the molecular engineering of membrane-mimetic biosystems because of the potential biological activities associated with natural membranes and their ability to self-organize as non-covalent aggregates. Phospholipids differing in chemical composition, saturation, and size have been utilized as building blocks in the design of structures of complex geometry, including lipid-based cylinders, cubes, and spheres. Surface-coupled bilayers have been produced by assembling a layer of closely packed hydrocarbon chains followed by exposure to either a dilute solution of emulsified lipids or unilamellar lipid vesicles (Spinke et al. [1992] Biophys. J. 63:1667; Florin et al. [1993] Biophys J. 64:375; Seifert et al. [1993] Biophys. J. 64:384). Langmuir-Blodgett techniques have also been used to construct supported bilayers via a process of controlled dipping of a substrate through an organic amphiphilic monolayer (Ulman [1991] An Introduction to Ultrathin Organic Films from Langmuir-Blodgett to Self-Assembly, New York: Academic Press). The overall significance of these design strategies lies in the ability to engineer surfaces in which the constituent members can be controlled, modified, and easily assembled with a high level of control over both order and chemistry. Of particular importance is the dialkyl moiety which facilitates the assembly of lipids with dissimilar head groups into surface structures of diverse biomolecular functionality and activity. Nonetheless, limited stability remains the major practical limitation of substrate supported membranes in which the constituent members are associated solely by non-covalent interactions.
In order to create robust surface structures, most membrane-mimetic systems for blood-contacting applications have been designed as copolymers containing the phosphorylcholine functional group in either side chains or, less frequently, the polymer backbone (Kojima et al. [1991] Biomaterials 12:121; Ueda, T. et al., [1992] Polym. J 24:1259; Ishihara, K. et al. [1995] Biomaterials 16:873; Campbell et al. [1994] ASAIO J. 40(3):M853; Chen et al. [1996] J. Appl. Polym. Sci. 60:455; and Yamada et al. [1995] JMS Pure Appl. Chem. A32:1723). While these materials have improved stability and promising blood-contacting properties have been reported, a number of limitations exist. In particular, the ability to engineer surface properties on a molecular level by taking advantage of the principle of self-organization intrinsic to amphiphilic molecules is lost. In addition, the ability to early incorporate diverse biomolecular functional groups into the membrane-mimetic surface is also lost.
The present invention provides the synthesis of stabilized, phosphorylcholine-containing polymeric surfaces by first attaching or incorporating a self-assembled acryloyloxy-containing phospholipid monolayer on an alkylated substrate and then subjecting the unit to in situ polymerization. This invention contemplates the production of the biomaterial through a process of assembly on a supported monolayer of modular surface design units, each possessing the desired physicochemical surface properties. Specifically, an example is provided of a generated surface which exhibits improved in vivo blood biocompatibility in a primate animal model.
The present invention also provides a new biomimetic approach for generating an ultra-thin organic barrier with the capacity for tailored transport and surface properties based upon a membrane-mimetic strategy. The extension of previous methodologies recently developed were utilized to produce a stable, lipid membrane-like bilayer on a hydrated alginate substrate. Marra, K. C. et al., Macromolecules 30:6483 (1997); Marra, K. C. et al., Langmuir 13:5697 (1997).
Transport characteristics and biocompatibility are critical membrane design properties for both the generation of controlled release drug delivery systems and capsules formulated as immunoisolation barriers for cell based therapy. Typically, membranes are produced with a variety of permeabilities by phase inversion processes whereby polymer precipitation time, polymer-diluent compatibility, and diluent concentration influences membrane porosity. In other systems, barriers can be created by a polyelectrolyte coacervation reaction and molecular weight cutoff (MWCO) is modulated by osmotic conditions, diluents, and the molecular weight distribution of the polycationic species. The utilization of multicomponent polyanionic polymer blends and the diffusion time of oligocationic species through precast blends of polyanionic polymers have also been shown to be important variables in the control of MWCO. Alginate-calcium chloride systems represent a third approach for generating semipermeable capsules and have been used to produce monodisperse, spherical, transparent beads at a high production rate. As a cell-compatible polysaccharide, alginate is an appealing polymer and, in addition, facilitates cryopreservation of the encapsulated cell. Control of transport properties, however, requires post-coating with a poly(amino acid), typically, poly-L-lysine or a derivative thereof. It is significant that transport characteristics are fundamentally governed, in all of these strategies, by the distribution of pore sizes created by thermodynamically driven physical processes.
Recent experiments have shown that non-covalently associated lipid bilayers can be deposited onto soft hydrated hydrophilic polymer cushions which in our view offers a route to barrier formation with enhanced control over both surface and transport properties. As described by Sackmann and coworkers (Kxc3xchner, M. et al., E. Biophys. J. 67:217 (1994); Elender, G. et al., E. Biosensors Bioelectronics 11:565 (1996)), a lipid monolayer is first formed on a dry dextran or polyacrylamide polymer film by vertical Langmuir-Blodgett dipping. The bilayer is completed after a second lipid layer is transferred using a Langmuir-Schxc3xa4efer technique and the formulated film stored underwater. In principle, functional reconstitution of membrane proteins including channels, transporters, and pores can be readily achieved. In addition, pores of well-defined size may be produced by suitable choice of template-forming guests in the membrane. The relatively low propensity toward biofouling is another appealing aspect of membrane-mimetic surfaces. As such, these systems have generated interest as a potential route to improved biocompatible biosensor design. Nonetheless, the stability of these supported membrane structures is limited since the lipid bilayer is not covalently coupled to the gel, nor are the self-associating lipid constituents stabilized in the two-dimensional plane by forces other than by van der Waal interactions.
The present invention provides a biocompatible biomaterial comprising a phospholipid or phospholipid derivative comprising various functional groups (e.g., lipid, peptide, sugar, etc.) having specific chemical properties which can function as a modular surface design unit to be incorporated or appended to a desired substrate on which it is then polymerized, thereby contributing new or specified biochemical characteristics to the polymerized and stabilized biomaterial. By first attaching a desired modular unit to a substrate (e.g., a polymer or a metal or derivatives thereof) and then carrying out in situ polymerization, the invention overcomes the disadvantages of unstable, non-permanent systems while providing the desired specificity of surface properties and biofunctionality in membrane-mimetic systems.
The present invention provides a biomaterial comprising a phospholipid or phospholipid derivative with a polymerizable monomeric group (e.g., acryloyloxy, methacryloyl, dienoyl, sorbyl, styryl, acrylamide, acrylonitrile, N-vinyl pyrrolidone, etc.). Such biomaterial phospholipid molecules form self-assembled monolayers that attach or absorb (e.g., through hydrophobic interactions, etc.) to a substrate whereon the polymerizable monomeric groups of the biomaterial phospholipid moieties are polymerized in situ. The biomaterial of the invention comprises two levels of attachment or cross reaction, i.e., (a) within the plane of phospholipid molecules, e.g., linking together different phospholipid alkyl chains, and (b) between planes, e.g., interdigitating chains between phospholipid monolayers and the substrate surface.
Biomaterials taught in the art are often covalently linked to a substrate. In the instant invention, a biomaterial is provided that is non-covalently affixed to a substrate, permitting a detachment of the polymerized biomaterial from the substrate or a replacement of one type of polymerized biomaterial by another type of biomaterial of the invention. The instant invention also contemplates biomaterials that are covalently attached to a substrate to fulfill a specific purpose or to meet a specific environmental condition. The biomaterials of the invention serve as specific modular surface design units. This concept of biomaterials composed of modular design units offers increased variability, versatility and flexibility not only with respect to choice of functional groups on a molecular or microscopic level (e.g., in the phospholipid functional groups such as phosphorylalkylamino groups, etc.) but also in the assembly of units into a layer on a macroscopic surface structure.
The instant invention provides particular exemplification of biocompatible biomaterial surfaces that includes, but is not limited by, (a) in situ polymerized phospholipids on solid alkylated surfaces of a self-assembled monolayer, e.g., octadecyltrichlorosilane (OTS) on glass, (b) in situ polymerized phospholipids on a polymer-supported monolayer of molecularly mobile alkyl chains, e.g, an amphiphilic, dialkyl-containing terpolymer bound to a gold-coated silicon wafer, and (c) in situ polymerized phospholipids onto a hydrogel (e.g., alginate) surface transformed into a hydrophobic surface by addition of an amphiphilic copolymer.
It is a particular object of the invention to provide a biocompatible biomaterial surface modular unit comprising a phospholipid moiety comprising a polymerizable monomeric group, e.g., an acryloyloxy group, methacryloyl, dienoyl, sorbyl, styryl, acrylamide, acrylonitrile, N-vinyl pyrrolidone, etc., which unit is attached or adsorbed or affixed to an alkylated substrate, and polymerized thereon in situ, in an amount and orientation effective to provide an improved nonthrombogenic surface relative to a substrate without the polymerizable monomeric group-containing phospholipid moiety attached thereto. The phospholipid moiety contains an alkyl amino group, e.g., a choline, ethanolamine or the like, and a phosphate polar group. In a preferred embodiment the biocompatible biomaterial has the structure (I): 
wherein R1 is a (C1-C30) alkyl group;
R2 is a (C1-C30) alkyl group;
m is 1-4;
n is 1-4;
Y is xe2x80x94CH2xe2x80x94CH2xe2x80x94+N(CH3)3 or xe2x80x94CH2xe2x80x94CH2xe2x80x94+NR3 
such that if R1 is attached to Z=xe2x80x94H, then R2 is attached to Z=
More preferably, the biocompatible biomaterial has the structure (I) wherein R1 is a (C12-C30) alkyl group; R2 is a (C8-C14) alkyl group; m is 1 and n is 1. In a preferred exemplification, the biocompatible material is 1-palmitoyl-2[12-(acryloyloxy)dodecanoyl]-sn-glycero-3-phosphorylcholine. The acrylate groups of the lipid molecules polymerize, forming a surface network in a two-dimensional plane.
The substrate of the invention includes, but is not limited to, an insoluble synthetic or natural, inorganic or organic material such as glass, silicon wafer, hydrogel (e.g., alginate, gelatin, collagen, polyhema, hydroxyethylmethacrylate, polyacrylamide, derivatives thereof, and the like) etc. The invention was particularly exemplified with alkylated substrates such as octadecyltrichlorosilane (OTS) coated glass, a self-assembling monolayer of an acylated octadecylmercaptan (e.g., ODT) on gold, octadecyl chains of an amphiphilic copolymer cast onto an alginate substrate, etc. A preferred substrate of the invention is exemplified by an amphiphilic dialkyl containing terpolymer bound to gold-coated silicon wafers. Thus, a preferred biomaterial of the invention comprises an acryloyloxy-containing phospholipid which is adsorbed to an amphiphilic polymer surface (a molecularly mobile alkylated surface extending from a polymer bonded to a substrate) and which is polymerized thereon.
It is an additional object of the invention to provide a biocompatible biomaterial that exhibits enhanced stability. In a particular example of this embodiment, a stabilized, phosphatidylcholine-containing polymeric surface was produced by in situ polymerization of 1-palmitoyl-2-[12-(acryloyloxy)dodecanoyl-sn-glycero-3-phosphorylcholine at a solid/liquid surface. The phospholipid monomer was synthesized, prepared as unilamellar vesicles, and fused onto close-packed octadecyl chains as part of an amphiphilic terpolymer. Free-radical polymerization was carried out according to the method of the invention. Contact angle measurements demonstrated that the polymerized lipid monolayer when supported by the amphiphilic terpolymer exhibited enhanced stability than when supported on a self-assembled monolayer of octadecyl mercaptan (ODT)-coated surface.
It is another object of the invention to provide a medical device, e.g., a shunt, stent or graft, etc., comprising an alkylated substrate on which is attached and polymerized a biocompatible biomaterial modular unit comprising a phospholipid moiety comprising an alkylamino group (preferably choline) linked to a polar phosphate group and a polymerizable monomeric group (preferably an acryloyloxy group).
It is a further object of the invention to provide a method of preparing a biocompatible biomaterial having improved biocompatibility. This biomaterial must comprise a polymerizable monomer (preferably an acryloyloxy group)-containing phospholipid moiety (preferably a phosphatidylcholine moiety) attached to, and polymerized in situ on, a substrate in an effective amount and orientation such that an improved nonthrombogenic surface is obtained relative to the substrate without the acryloyloxy-containing phospholipid moiety. The method for preparing a biocompatible biomaterial of the invention comprises the steps of:
(a) synthesizing a phosphorylalkylamino-containing phospholipid comprising a polymerizable monomeric group;
(b) preparing lipid vesicles from said phospholipid of step (a);
(c) attaching or adsorbing said vesicles of step (b) onto a substrate; and
(d) exposing the attached or adsorbed vesicles of step (c) to an initiator of polymerization such that the phospholipid undergoes in situ polymerization, forming a biopolymer or biomaterial having improved biocompatibility.
Improved biocompatibility is assessed according to the invention in a mammalian model in vivo or in an in vitro assay as a condition exhibiting decreased thrombogenicity or coagulation.
In further embodiments, the biomaterial of the invention is prepared to possess improved stability of a polymerized lipid monolayer at a solid/liquid interface. Improved stability is provided by utilizing a substrate comprising long chain acyl chains extending from an amphiphilic polymer surface. In a particular embodiment, the invention was exemplified by in situ polymerized phospholipid on an amphiphilic, dialkyl-containing terpolymer.
In other embodiments, a stabilized, phosphatidylcholine-containing polymeric surface was produced by in situ polymerization of 1-palmitoyl-1-[12-(acryloyloxy)dodecanoyl]-sn-glycero-3-phosphoryl-choline at a solid/liquid interface. The phospholipid monomer was synthesized, prepared as unilamellar vesicles, and fused onto close-packed octadecyl chains as part of an amphiphilic copolymer. The copolymer was cast onto a hydrogel, e.g., alginate, thus transforming a hydrophilic surface into a hydrophobic surface. Free-radical polymerization of the phospholipid vesicles was carried;out in aqueous solution. The supported monolayer displayed properties consistent with theoretical predictions for lipid membrane. The present invention provides a method for the transformation of a hydrophobic surface into a hydrophilic antithrombogenic surface.
In various exemplifications of the invention, free-radical polymerizations were carried out using a water-soluble initiator, e.g., 2,2xe2x80x2-azobis(2-methylpropionamidine) dihydrochloride (AAPD), or an oil-soluble initiator, e.g., 2,2xe2x80x2-azobisisobutyronitrile (AIBN).
It is yet another object of the invention to provide a biopolymer or biomaterial, prepared by the method of the invention, that demonstrates acceptable stability under static conditions in water and air, as well as in the presence of a high shear flow environment. In addition, this biopolymer or biomaterial, prepared by the method of the invention, exhibits blood compatibility in a mammalian model system. In a particular embodiment of the invention, an arteriovenous shunt prepared with a biomaterial of the invention, when placed in a baboon, revealed minimal platelet deposition.